1. Field of the Invention
The present invention relates to a method of using reagents as calibrators (standards) and/or controls in diagnostic assays and kits. More specifically, one or more of these reagents may be used in place of seropositive plasma or serum in the production of calibrators and/or controls for diagnostic assays and kits designed to qualitatively or quantitatively measure antibodies specific for a desired ligand. The reagents themselves bind specifically to a predetermined ligand, contain one or more antibody constant region epitopes, and are homogeneous in specificity and affinity. The reagents may be produced by the use of hybridoma and/or recombinant DNA technology.
2. Background Information
Antibodies are multidomain proteins composed of two identical light (L) and two identical heavy (H) polypeptide chains, linked together by disulfide bonds (FIG. 1). The amino terminal domain of both the L and H chains exhibits considerable diversity in amino acid sequence and conformation and is referred to as the variable (V) region. Residing within each V region are three segments of exceptional variability, referred to as hypervariable regions or complementarity-determining regions (CDRs), that form the ligand binding pocket. The other domains of both the L and H chains constitute the constant regions. The constant regions are not involved in ligand binding and exhibit more limited variation. Constant regions are species specific and can be divided into various classes and subclasses based on differences in the heavy chain constant region including size, charge, amino acid composition, glycosylation and biological function (Carayannopoulos, L. and Capra, J. D. Structure and Function of Immunoglobulins, In Fundamental Immunology, 3rd edition, Paul W. E. ed., Raven Press Ltd., New York, pgs. 283-314 (1993)).
The immune system generates a remarkably diverse repertoire of antibody molecules capable of recognizing virtually any substance. The primary antibody response induced by challenge with antigen (i.e., any substance capable of eliciting an immune response, for example, proteins, carbohydrates, nucleic acids, lipids, or hapten conjugated to a carrier) results in the production of antibodies predominantly of the IgM class. This response is polyclonal in nature, as a heterogeneous mixture of antibodies against different epitopes of the antigen is produced. Subsequent or prolonged challenge with the same antigen leads to a secondary response characterized by significantly greater titers of antibody than that seen in the primary response that are generally higher in affinity (measure of the binding strength between an epitope and the antibody combining site) and composed almost entirely of the IgG class. The specific IgM titer generally wanes more rapidly than the specific IgG titer. As a result, monitoring the class of specific antibody present in serum or other biological fluids provides an indicator of the individual's immune status to a specific antigen (e.g., infectious agents). Antibody class can also be of clinical relevance in cases of autoimmunity and for monitoring Type I hypersensitivity (allergic responses) associated with production of IgE class antibodies (Roitt, I. ed., Immunology, Gower Medical Publishing Ltd., London, England (1985); Paul W. E. ed., Fundamental Immunology, 3rd edition, Raven Press Ltd., New York (1993)).
Irrespective of class, antibody molecules bind to ligands with high affinity and specificity (the ability to discriminate between the epitope to which it is directed and any other epitope) making them ideal immunodiagnostic reagents. Immunoassays provide a rapid and sensitive method to monitor for infectious agents, physiological function, allergy, autoimmunity, cancer, pharmaceuticals, and drugs of abuse. Manual and automated immunoassays have been designed to measure the antibody response in general, antibody to a specific antigen, and diagnostically relevant antigens or haptens. Heterologous immunoassays generally consist of multiple reaction steps and ultimately require separation of the immune complexed reactant from the free reactants to obtain the test result. In contrast, homogeneous immunoassays, are solution-phase systems that do not require the separation of complexed reactant from free reactants. Immunoassays have been developed with many different formats, but can be divided into two main classes: (1) competitive assays, and (2) non-competitive assays (e.g. immunometric, sandwich). For heterologous immunoassays of both classes, solid-phase biochemistry for separation of bound and free reactants has proven revolutionary. Antibody or antigen reagents can be covalently or non-covalently (e.g. ionic, hydrophobic) attached to the solid phase. Linking agents for covalent attachment are known and may be part of the solid phase or derivatized to it prior to coating. Examples of solid phases used in immunoassays are, porous and non-porous materials, latex particles, magnetic particles, microparticles, beads, membranes, microtiter wells and plastic tubes. The choice of solid phase material and method of labeling the antigen or antibody reagent is determined based on desired assay format performance characteristics. For some immunoassays, no label is required. For example, if the antigen is on a detectable particle such as a red blood cell, reactivity can be established based on agglutination. Alternatively, antigen-antibody reaction may result in a visible change (e.g., radial immunodiffusion). In most cases, one of the antibody or antigen reagents used in an immunoassay is attached to a signal generating compound or "label". This signal generating compound or "label" is in itself detectable or may be reacted with one or more additional compounds to generate a detectable product. Examples of signal generating compounds include chromogens, radioisotopes (e.g. .sup.125 I, .sup.131 I, .sup.32 P, .sup.3 H, .sup.35 S, and .sup.14 C), fluorescent compounds (e.g. fluorescein, rhodamine), chemiluminescent compounds, particles (visible or fluorescent), nucleic acids, complexing agents, or catalysts such as enzymes (e.g. alkaline phosphatase, acid phoshatase, horseradish peroxidase, beta-galactosidase, and ribonuclease). In the case of enzyme use, addition of chromo-, fluoro- or lumo-genic substrate results in generation of a detectable signal. Other detection systems such as time-resolved fluorescence, internal-reflection fluorescence, amplification (e.g. polymerase chain reaction) and Raman spectroscopy are also useful.
Immunoassays have been developed to monitor biological fluids (e.g., plasma, serum, cerebrospinal fluid, saliva, tears, nasal washes, or aqueous extracts of tissues and cells) for the presence of antibody specific for an antigen of interest (e.g. infectious agent, autoantigen, allergen). In many cases, these specific antibody immunoassays have been designed to be antibody class or subclass specific. There are two general formats commonly utilized to monitor specific antibody in humans: (1) antigen is presented on a solid phase, the human biological fluid containing specific antibodies is allowed to react with the antigen, and then antibody bound to antigen is detected with an anti-human antibody coupled to a signal generating compound and (2) an anti-human antibody is bound to the solid phase, the human biological fluid containing specific antibodies is allowed to react with the antibody, and then antigen attached to a signal generating compound is added to detect specific antibody. In both formats, the anti-human antibody reagent can be polyclonal or monoclonal. Moreover, the anti-human antibody reagent may recognize all antibody classes, or alternatively, be specific for a particular class or subclass of antibody, depending upon the intended purpose of the assay. The reactivity of this reagent reflects the spectrum of antibodies of which it is composed and the particular antibody constant region epitopes to which they bind. Constant region epitopes are antigenic determinants to which an antibody response can be generated. Examples of constant region epitopes include: species invariant epitopes (class or subclass specific epitopes), and allotypic epitopes (present on some, but not all members of a species). Methods for manufacturing and testing of anti-human antibody reagents that bind to constant region epitopes are well known to the art. Assays for monitoring of specific antibody response in other species could be designed in an analogous manner by one skilled in the art.
Immunoassays designed to detect specific antibody provide a measure of antibody activity. This may be referred to as antibody titer, e.g. mid-point or end-point titer, or expressed in units (activity or gravimetric) relative to a reference standard. Immunoassays and kits typically include one or more components containing the specific antibody being measured that function as calibrators (standards) and/or positive control. Calibrators (standards) are used to establish calibration (standard) curves for interpolation of antibody concentration, or alternatively, a single calibrator may be used near the positive/negative cutoff. The positive control is used to establish assay performance characteristics and is a useful indicator of the integrity of the reagents. In addition, immunoassays and kits to detect specific antibody generally include a negative control, such as serum or plasma, that contains no antibody reactive with the antigen of interest. Preferably, the calibrators and positive controls are prepared with the specific antibody being measured or a material chemically similar to it. Ideally the calibrator(s) and controls are manufactured to interact with the other assay components in a manner analogous to the test analyte (specific antibody). Calibrators and positive controls are generally manufactured by spiking known quantities of specific antibody derived from seropositive plasma or serum (polyclonal antibody) into the negative control reagent. Often times multiple calibrators are included that contain varying concentrations of specific antibody that span the range of concentrations the assay is designed to measure. Quantitative immunoassays may include up to 8 calibrators (standards) to establish a calibration (standard) curve from which results can be interpolated. The quantity of specific antibody assigned to the calibrator(s) and control(s) is standardized against primary reference standards. In the case of antibody activity, the reference standard is often established using individual or pooled sera that has been characterized empirically for characteristics such as quantity, quality, and specificity. The reference standard may be assigned relative units of activity or be a gravimetric quantity of antibody. For qualitative immunoassays, a single calibrator (standard) may be used that is set near the positive/negative cutoff. Some manufacturer's refer to the single calibrator (standard) as an index calibrator (Voller, A. et. al., Immunoassays for the 80s, University Park Press, Baltimore (1981); Albertini, A. and Ekins, R., eds., Monoclonal Antibodies and Developments in Immunoassay, Elsevier/North-Holland Biomedical Press, New York (1981); Butler, J., ed., Immunochemistry of Solid-Phase Immunoassay, CRC Press, Boca Raton (1991)).
Two examples of an immunometric antibody-capture based immunoassay are the IMx Toxo IgM and Toxo IgG (FIG. 2) antibody assays manufactured by Abbott Laboratories. Both assays are automated Microparticle Enzyme Immunoassay (MEIA) which measure antibodies to Toxoplasma gondii (T. gondii) in human serum or plasma (Safford, J. W. et. al., J. Clin. Pathol. 44:238-242 (1991)). One assay qualitatively measures IgM antibodies, indicative of recent exposure or acute infection, and the other assay quantitatively measures IgG, indicative of chronic or past infection. T. gondii, an obligate intracellular parasite infecting adults often asymptomatically, can lead to serious consequences for the fetus due to transplacental transmission which occurs during acute acquired maternal infection (Remington, J. S. and Krahenbuhl J. L., Comprehensive Immunology (A. J. Nahmias and O'Reilly. Eds.) pgs. 327-371. Plenum, New York/London (1982); Remington, J. S., Intrauterine Infections: Birth defects Origin. Ser 4:47-56. The National Foundation of the March of Dimes, New York (1968)). Determination of maternal immune status by testing for the presence of T. gondii specific IgM or IgG antibodies can aid in the determination of pregnancies at risk and in the case of seronegative individuals allows one to monitor for seroconversion that would be indicative of acute infection (Desmonts, G. and Couvreur J., New Engl. J. Med. 290:1110-1116 (1974); McCabe, R. and Remington J. S., New Engl. J. Med. 318:313-317 (1988); Sibalic, D. et. al., Gynecol. Obstet. Invest. 36:91-95 (1993)). These assays use microparticles coated with T. gondii antigens as the solid phase. Specimen is added to the coated microparticles to allow antibodies specific for T. gondii to bind. Subsequently, an alkaline phosphatase conjugated anti-human IgM (or anti-human IgG) is added that binds specifically to IgM (or IgG) class antibodies complexed to the T. gondii antigens. Following addition of a suitable substrate, the rate of enzyme-catalyzed turnover is monitored based on fluorescence.
The calibrators and positive controls for the IgM and IgG anti-T. gondii assays are prepared from plasma collected from human donors reactive to T. gondii. High titer plasma or serum has traditionally served as a source of controls and/or calibrators (standards) in diagnostic assays and kits designed to monitor for the presence of human antibodies specific for a given antigen. These include tests to detect antibodies to human immunodeficiency virus-1, human immunodeficiency virus-2, human T-cell leukemia virus-1, human T-cell leukemia virus-2, cytomegalovirus, hepatitis A virus, hepatitis B virus, hepatitis C virus, hepatitis D virus, hepatitis E virus, respiratory syncytial virus, Rubella virus, Toxoplasma gondii, Trypanosoma cruzi, Cryptococcus neoformans, Histoplasma capsulatum, Helicobacter pylon, and Streptococcus pyogenes. Use of human seropositive plasma or serum for the manufacture of calibrators (standards) and/or controls has several significant drawbacks, including: (1) the increasing difficulty of sourcing large volumes of plasma or serum with high titer, high specificity, and that lack antibodies to other infectious agents, (2) considerable lot-to-lot variability over time with respect to titer and specificity which impacts the performance of the assay, (3) inherent limitations with respect to characterization of an antisera due to its polyclonal nature (heterogeneous in antibody class, specificity and affinity), and (4) cost. In fact, it is conceivable that, in some cases, it may be impossible to maintain production of calibrators and/or controls that are based on seropositive human plasma due to sourcing issues. It can be especially difficult to find high titer sources of IgM antibody as these are generally obtained from acutely infected individuals. For example, sourcing of IgM reactive to Rubella in the U.S. is difficult due to the successful vaccination program. As sourcing of suitable seropositive plasma or serum becomes more difficult, the costs related to manufacturing of calibrators and controls increase, and this economic burden is ultimately passed on to the patient. An alternative method for manufacture of calibrators (standards) and/or positive controls for diagnostic assays and kits designed to monitor levels of specific antibody would represent a significant advance.
Genetically engineered antibodies, such as mouse:human chimeric antibodies, can be used as quality control reagents for specificity testing of anti-human antibody conjugates and for quality control of human total immunoglobulin immunoassays. It was further proposed that these chimeric antibodies may be useful reagents for quantitation of specific antibody in reference standards. The chimeric antibodies would be used to establish a heterologous dose-response curve to interpolate the amount of antibody in a reference standard specific for a different antigen (Hamilton, R. G., Ann. Biol. Clin. 48:473-477 (1990); Butler, J. E. and Hamilton, R. G., In Immunochemistry of Solid-phase Immunoassay, Butler, J. E. ed., CRC Press, Boca Raton, pgs. 173-198 (1991)). The term "heterologous" indicates that the antigen for which the chimeric antibodies are specific is defined, but unrelated to the antigen to which specific antibody is being monitored.
The present invention differs from the aforementioned in two important ways: (1) the proposed reagents are intended to be used, as a substitute for seropositive plasma or serum, in the manufacture of calibrators (standards) and/or positive controls for immunoassays and kits designed to monitor antigen specific antibody responses, and (2) the proposed reagents bind to the same or "homologous" antigen to that which the specific antibody being measured binds. Use of reagents which bind to the homologous antigen is preferable in that the calibrator, positive control, and test specimen are reacted to the same antigen under identical conditions, providing a more realistic measure of specific antibody activity, and has the added advantage of allowing one to monitor the integrity of the test antigen at the time the assay is run.
One alternative source of material for the manufacture of positive controls in immunoassays designed to detect specific human antibody is non-human immune antibody that reacts with an anti-human antibody (U.S. Pat. No. 5,008,183). Use of non-human immune (polyclonal) sera has several drawbacks including: (a) lot-to-lot variability over time with respect to antibody class composition, titer, specificity and affinity that can impact assay performance; (b) difficulty in characterization due to its polyclonal composition (e.g. heterogeneous affinity and specificity); (c) limited supply; and (d) in the case of infectious agents, potential biohazard if live organisms are used to immunize.
An alternative source of material for the manufacture of calibrators and controls for IgM assays is a composite antibody of a nonspecific IgM immunoglobulin moiety covalently linked to a specific non-IgM antibody moiety (U.S. Pat. No. 5,478,753). Use of immune sera to produce the composite antibody has all of the inherent drawbacks outlined above. In addition, one must source and purify both the immune and non-immune moieties used to construct the composite antibody. Moreover, the chemically crosslinked products would be heterogeneous with respect to the number and location of the attached non-immune antibody moieties. In fact, attachment near the binding site may result in steric interference of antigen binding by the specific antibody.
Use of the reagents described in the present invention, that bind to ligand, contain one or more constant region epitopes, and are homogeneous in specificity and affinity (i.e., all molecules are uniform with respect to the properties of specificity and affinity), circumvent all of the problems associated with using an immune sera in manufacture of calibrators and positive controls. Furthermore, since the constant region epitopes are an integral part of the reagent (i.e., directly fused to the ligand binding domain) a more uniform composition is obtained. Moreover, the present reagents can be readily and reproducibly generated in virtually unlimited quantities and are also useful for quantitating, and monitoring the integrity of, antigen used in the assay.